Mechanism and function of Cdc123 mediated eIF2 assembly

The eukaryotic initiation factor 2 (eIF2) is a protein complex which is conserved among all eukaryotes and has a homolog in archaea, called archaeal initiation factor 2 (aIF2). It fulfills essential functions in protein synthesis and is made up of three subunits (α, β and γ). Together with GTP and the initiator tRNA Met-tRNAiMet (tRNAi), eIF2 forms the ternary complex (TC). eIF2 leads tRNAi to the ribosomal 40S subunit and scans the mRNA in collaboration with other initiation factors. Upon binding of tRNAi to a start codon, γ-phosphate of the GTP and subsequently all initiation factors are released (Algire et al., 2005). The ribosomal subunits join to form an active 80S ribosome and elongation phase, in which the peptide chain is synthesized, commences. eIF2 is involved in selection of the right start codon and important for the accuracy of protein synthesis. It is a hub for central regulational mechanisms, which adjust cell metabolism under conditions of stress. Amino acid starvation, unfolded protein stress and other stress factors activate certain kinases, which phosphorylate eIF2α, reduce TC activity and thus protein synthesis rate (Pakos-Zebrucka et al., 2016). On a molecular level, defects in eIF2 lead to inefficient protein synthesis and inaccurate selection of start codons, hence affecting cell growth and increasing susceptibility to exogenous stressors. In humans, several rare mutations of eIF2γ are known which negatively affect the activity of eIF2 and lead to organ dysfunction and neuronal defects in particular. The molecular cause is lowered TC activity, since binding of tRNAi and assembly of trimeric eIF2 are affected (Borck et al., 2012; Young-Baird et al., 2019c). This work pays particular attention to the assembly mechanism of eIF2. To date, no dedicated scaffold protein or chaperone that necessary for aIF2 assembly in archaea is known. In eukaryotes however, previous studies established that the protein Cdc123 is indispensable for assembly of the the α-, β- and γ-subunits to active trimeric eIF2 (Perzlmaier et al., 2013). Meanwhile, little is known about the order of assembly and the individual functions of each subunit as well as Cdc123. However, the mechanism of complex formation is of great interest to understand molecular causes of eIF2-related hereditary defects. Here, detailed studies of eIF2 subunit interactions and eIF2-Cdc123 interactions will be presented. The majority of experiments were performed in the budding yeast S. cerevisiae. Thanks to its ease of handling and fast replication, it is an ideal model organism for fundamental cellular processes. The high conservation of the translation process in general and eIF2 in particular allow for conclusions about the respective processes in human cells. In yeast, the endogenous proteins and, by heterologous expression, the human proteins were investigated. Some results were verified in human cell culture. At the end, a model for stepwise assembly of eIF2 will be presented and individual roles of each eIF2 subunit and Cdc123 will be discussed

Activated PI3Kδ syndrome – reviewing challenges in diagnosis and treatment

Activated PI3Kδ syndrome (APDS) is a rare inborn error of immunity (IEI) characterized primarily by frequent infections, lymphoproliferation and autoimmunity. Since its initial description in 2013, APDS has become part of the growing group of nearly 500 IEIs affecting various components of the immune system. The two subtypes of APDS - APDS1 and APDS2 - are caused by variants in the PIK3CD and PIK3R1 genes, respectively. Due to the rarity of the disease and the heterogeneous clinical picture, many patients are not diagnosed until years after symptom onset. Another challenge is the large number of PIK3CD and PIK3R1 variants whose functional significance for developing APDS is inconclusive. Treatment of APDS has so far been mostly symptom-oriented with immunoglobulin replacement therapy, immunosuppressive therapies and antibiotic or antiviral prophylaxes. Additionally, allogeneic stem cell transplantation as well as new targeted therapies are options targeting the root cause that may improve patients’ quality of life and life expectancy. However, the clinical course of the disease is difficult to predict which complicates the choice of appropriate therapies. This review article discusses diagnostic procedures and current and future treatment options, and highlights the difficulties that physicians, patients and their caretakers face in managing this complex disease. This article is based on cohort studies, the German and US guidelines on the management of primary immunodeficiencies as well as on published experience with diagnosis and compiled treatment experience for APDS

Stepwise assembly of the eukaryotic translation initiation factor 2 complex

The eukaryotic translation initiation factor 2 (eIF2) has key functions in the initiation step of protein synthesis. eIF2 guides the initiator tRNA to the ribosome, participates in scanning of the mRNA molecule, supports selection of the start codon, and modulates the translation of mRNAs in response to stress. eIF2 comprises a heterotrimeric complex whose assembly depends on the ATP-grasp protein Cdc123. Mutations of the eIF2γ subunit that compromise eIF2 complex formation cause severe neurological disease in humans. To this date, however, details about the assembly mechanism, step order, and the individual functions of eIF2 subunits remain unclear. Here, we quantified assembly intermediates and studied the behavior of various binding site mutants in budding yeast. Based on these data, we present a model in which a Cdc123-mediated conformational change in eIF2γ exposes binding sites for eIF2α and eIF2β subunits. Contrary to an earlier hypothesis, we found that the associations of eIF2α and eIF2β with the γ-subunit are independent of each other, but the resulting heterodimers are nonfunctional and fail to bind the guanosine exchange factor eIF2B. In addition, levels of eIF2α influence the rate of eIF2 assembly. By binding to eIF2γ, eIF2α displaces Cdc123 and thereby completes the assembly process. Experiments in human cell culture indicate that the mechanism of eIF2 assembly is conserved between yeast and humans. This study sheds light on an essential step in eukaryotic translation initiation, the dysfunction of which is linked to human disease

Future Directions in the Diagnosis and Treatment of APDS and IEI: a Survey of German IEI Centers

IntroductionThe diagnosis and treatment of inborn errors of immunity (IEI) is a major challenge as the individual conditions are rare and often characterized by a variety of symptoms, which are often non disease-specific. Ideally, patients are treated in dedicated centers by physicians who specialize in the management of primary immune disorders. In this study, we used the example of Activated PI3Kδ syndrome (APDS), a rare IEI with an estimated prevalence of 1:1,000,000. We conducted surveys by questionnaire and interviewed physicians at different IEI centers in Germany.MethodsWe queried structural aspects of IEI care in Germany, diagnostic procedures in IEI care (including molecular diagnostics), distribution of APDS patients, APDS symptoms and severity, treatment algorithms in APDS, the role of stem cell transplantation and targeted therapies in IEI with focus on APDS. We were especially interested in how genetic diagnostics may influence treatment decisions, e.g. with regard to targeted therapies.Results/discussionMost centers care for both pediatric and adult patients. A total of 28 APDS patients are currently being treated at the centers we surveyed. Patient journeys vary considerably, as does severity of disease. Genetic diagnosis continues to gain importance - whole genome sequencing is likely to become routine in IEI in the next few years. According to the experts interviewed, stem cell transplantation and - with new molecules being approved - targeted therapies, will gain in importance for the treatment of APDS and IEI in general

Solar storms may trigger sperm whale strandings: explanation approaches for multiple strandings in the North Sea in 2016

The Earth's atmosphere and the Earth's magnetic field protects local life by shielding us against Solar particle flows, just like the sun's magnetic field deflects cosmic particle radiation. Generally, magnetic fields can affect terrestrial life such as migrating animals. Thus, terrestrial life is connected to astronomical interrelations between different magnetic fields, particle flows and radiation. Mass strandings of whales have often been documented, but their causes and underlying mechanisms remain unclear. We investigated the possible reasons for this phenomenon based on a series of strandings of 29 male, mostly bachelor, sperm whales (Physeter macrocephalus) in the southern North Sea in early 2016. Whales’ magnetic sense may play an important role in orientation and migration, and strandings may thus be triggered by geomagnetic storms. This approach is supported by the following: (1) disruptions of the Earth's magnetic field by Solar storms can last about 1 day and lead to short-term magnetic latitude changes corresponding to shifts of up to 460 km; (2) many of these disruptions are of a similar magnitude to more permanent geomagnetic anomalies; (3) geomagnetic anomalies in the area north of the North Sea are 50–150 km in diameter; and (4) sperm whales swim about 100 km day-1, and may thus be unable to distinguish between these phenomena. Sperm whales spend their early, non-breeding years in lower latitudes, where magnetic disruptions by the sun are weak and thus lack experience of this phenomenon. ‘Naïve’ whales may therefore become disoriented in the southern Norwegian Sea as a result of failing to adopt alternative navigation systems in time and becoming stranded in the shallow North Sea

Seawater carbonate chemistry and gene expression in the gills of juvenile turbot (Psetta maxima)

The data sets stem from a study which aimed to investigate the impact of chronic severe hypercapnia on the cellular stress response in the gills of juvenile turbot (Psetta maxima). The experiment was conducted in the so called respirometer, a recirculating aquaculture respirometer system (RARS) which allowed the persistent, stable manipulation of carbon dioxide concentrations. Juvenile turbot (Psetta maxima) where subjected to different levels of environmental hypercapnia (low: ~3000 μatm, medium: ~15000 μatm, high: ~25000 μatm). After eight weeks, fish where sacrificed and gill samples were taken for further histological and transcriptional analysis. For histological investigation of the gills, samples where fixed in 4% phosphate-buffered formalin and examined via a microscope equipped with a digital camera. In order to investigate changes in the transcriptome, a fraction of the gill samples was stored in RNAlater. Relative gene expression analysis was conducted via RT-qPCR, using a Fluidigm chip system. Calibrated normalized relative quantities (CNRQs) where assessed with the QBASE software. This included the evaluation of the most suitable combination of reference genes with the help of the GeNorm algorithm